The activation of NF-kappaB by a wide variety of stimuli has been strongly implicated in tumorigenesis and clinical resistance. Abrogation of NF-kappaB signaling has been observed to effectively sensitize cancer cells to chemotherapeutics in the laboratory; however, the development of clinical NF-kappaB inhibitors has proven difficult. Due to the extensive role of NF-kappaB in mediating basic physiological processes such as immune and inflammatory response, abolishing NF-kappaB activation leads to toxic side effects. This proposal aims to attenuate NF-kappaB signaling by disrupting the recruitment of the critical NF-kappaB inhibitor of kappaB kinase (IKK) complex without disrupting its catalytic activity. The IKK complex is composed of three subunits, NEMO/IKKy (NF-kappaB essential modulator) which acts as a scaffolding molecule and two catalytic subunits IKKalpha and IKKbeta. NEMO's primary function in canonical NF-kappaB activation is to bind K63- or linearly-linked polyubiquitin chains to recruit the IKK complex to activated receptors. Recently, our laboratory has discovered a drug, withaferin a, that can modify NEMO to alter its preferences for binding K48-linked ubiquitin chains. Therefore, we hypothesize that NEMO may contain a novel ubiquitin binding domain that is capable of recognizing K48-linked ubiquitin chains which can be targeted to attenuate NF-kappaB activation. The following aims will test our hypothesis using two complementary approaches. Aim1: In a biochemical approach, the novel ubiquitin-binding domain (UBD) of NEMO will be mapped to the amino acid level and assessed for its ability to bind K48-linked ubiquitin chains in vitro. Aim2: In the functional approach, a genetic complementation screen will be conducted to assess the ability of NEMO mutants to attenuate NF-kappaB activation at the level of recruitment to activated receptor complexes in vivo. Moreover, this aim will identify novel molecules that regulate NEMO's interaction with K48-linked ubiquitin chains. We believe that proposed research will make two major contributions to the scientific community. First, the described research will enhance and expand our knowledge of the role of ubiquitin in NF-kappaB signaling pathways. Secondly, understanding the signaling mechanisms that are involved in cancer cell survival will reveal rational therapeutic targets for the development of novel anti-cancer drugs.